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Found 3 matching solutions for this experiment
| Upstream tips |
Protocol tips |
Downstream tips |
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Staining of surface molecules was always performed by incubating cells with specific mAbs or the respective isotype control for 15 min at 4 °C in the dark and at a concentration as recommended by the manufacturer. Then, cells were washed with MACS‐buffer (see above) and fixed with 1% formaldehyde (BÜFA Chemicals GmbH&Co.KG, Hude, Germany). In case of biotin‐conjugated mAbs, cells were incubated with Streptavidin‐PerCp/Cy5.5 at a final concentration of 1:100 for 15 min at 4 °C followed by a second washing step before fixation. |
When combining extra‐cellular and intra‐cellular stainings, first the surface staining was performed. After the last washing step, cells were treated with freshly prepared fixation/permeabilization solution for 10 min at room temperature in the dark, washed with permeabilization buffer (both from eBioscience) and followed by incubation with fluorochrom‐conjugated specific mAbs diluted in permeabilization buffer for 10 min at room temperature in the dark. After washing with permeabilization buffer, cells were finally fixed with 1% formaldehyde. Flow cytometric analysis was always performed with a FACS Calibur (BD Biosciences, Heidelberg, Germany) equipped with an argon laser. |
| Protocol tips |
| Staining of surface molecules was always performed by incubating cells with specific mAbs or the respective isotype control for 15 min at 4 °C in the dark and at a concentration as recommended by the manufacturer. Then, cells were washed with MACS‐buffer (see above) and fixed with 1% formaldehyde (BÜFA Chemicals GmbH&Co.KG, Hude, Germany). In case of biotin‐conjugated mAbs, cells were incubated with Streptavidin‐PerCp/Cy5.5 at a final concentration of 1:100 for 15 min at 4 °C followed by a second washing step before fixation. |
| Downstream tips |
| When combining extra‐cellular and intra‐cellular stainings, first the surface staining was performed. After the last washing step, cells were treated with freshly prepared fixation/permeabilization solution for 10 min at room temperature in the dark, washed with permeabilization buffer (both from eBioscience) and followed by incubation with fluorochrom‐conjugated specific mAbs diluted in permeabilization buffer for 10 min at room temperature in the dark. After washing with permeabilization buffer, cells were finally fixed with 1% formaldehyde. Flow cytometric analysis was always performed with a FACS Calibur (BD Biosciences, Heidelberg, Germany) equipped with an argon laser. |
| Upstream tips |
Protocol tips |
Downstream tips |
|
Staining of surface molecules was always performed by incubating cells with specific mAbs or the respective isotype control for 15 min at 4 °C in the dark and at a concentration as recommended by the manufacturer. Then, cells were washed with MACS‐buffer (see above) and fixed with 1% formaldehyde (BÜFA Chemicals GmbH&Co.KG, Hude, Germany). In case of biotin‐conjugated mAbs, cells were incubated with Streptavidin‐PerCp/Cy5.5 at a final concentration of 1:100 for 15 min at 4 °C followed by a second washing step before fixation. |
When combining extra‐cellular and intra‐cellular stainings, first the surface staining was performed. After the last washing step, cells were treated with freshly prepared fixation/permeabilization solution for 10 min at room temperature in the dark, washed with permeabilization buffer (both from eBioscience) and followed by incubation with fluorochrom‐conjugated specific mAbs diluted in permeabilization buffer for 10 min at room temperature in the dark. After washing with permeabilization buffer, cells were finally fixed with 1% formaldehyde. Flow cytometric analysis was always performed with a FACS Calibur (BD Biosciences, Heidelberg, Germany) equipped with an argon laser. |
| Protocol tips |
| Staining of surface molecules was always performed by incubating cells with specific mAbs or the respective isotype control for 15 min at 4 °C in the dark and at a concentration as recommended by the manufacturer. Then, cells were washed with MACS‐buffer (see above) and fixed with 1% formaldehyde (BÜFA Chemicals GmbH&Co.KG, Hude, Germany). In case of biotin‐conjugated mAbs, cells were incubated with Streptavidin‐PerCp/Cy5.5 at a final concentration of 1:100 for 15 min at 4 °C followed by a second washing step before fixation. |
| Downstream tips |
| When combining extra‐cellular and intra‐cellular stainings, first the surface staining was performed. After the last washing step, cells were treated with freshly prepared fixation/permeabilization solution for 10 min at room temperature in the dark, washed with permeabilization buffer (both from eBioscience) and followed by incubation with fluorochrom‐conjugated specific mAbs diluted in permeabilization buffer for 10 min at room temperature in the dark. After washing with permeabilization buffer, cells were finally fixed with 1% formaldehyde. Flow cytometric analysis was always performed with a FACS Calibur (BD Biosciences, Heidelberg, Germany) equipped with an argon laser. |
| Upstream tips |
Protocol tips |
Downstream tips |
|
. In addition, Streptavidin‐PerCP/Cy5.5 (Biolegend) was used as secondary staining reagent. Staining of surface molecules was always performed by incubating cells with specific mAbs or the respective isotype control for 15 min at 4 °C in the dark and at a concentration as recommended by the manufacturer. Then, cells were washed with MACS‐buffer (see above) and fixed with 1% formaldehyde (BÜFA Chemicals GmbH&Co.KG, Hude, Germany) |
When combining extra‐cellular and intra‐cellular stainings, first the surface staining was performed. After the last washing step, cells were treated with freshly prepared fixation/permeabilization solution for 10 min at room temperature in the dark, washed with permeabilization buffer (both from eBioscience) and followed by incubation with fluorochrom‐conjugated specific mAbs diluted in permeabilization buffer for 10 min at room temperature in the dark. After washing with permeabilization buffer, cells were finally fixed with 1% formaldehyde. Flow cytometric analysis was always performed with a FACS Calibur (BD Biosciences, Heidelberg, Germany) equipped with an argon laser. |
| Protocol tips |
| . In addition, Streptavidin‐PerCP/Cy5.5 (Biolegend) was used as secondary staining reagent. Staining of surface molecules was always performed by incubating cells with specific mAbs or the respective isotype control for 15 min at 4 °C in the dark and at a concentration as recommended by the manufacturer. Then, cells were washed with MACS‐buffer (see above) and fixed with 1% formaldehyde (BÜFA Chemicals GmbH&Co.KG, Hude, Germany) |
| Downstream tips |
| When combining extra‐cellular and intra‐cellular stainings, first the surface staining was performed. After the last washing step, cells were treated with freshly prepared fixation/permeabilization solution for 10 min at room temperature in the dark, washed with permeabilization buffer (both from eBioscience) and followed by incubation with fluorochrom‐conjugated specific mAbs diluted in permeabilization buffer for 10 min at room temperature in the dark. After washing with permeabilization buffer, cells were finally fixed with 1% formaldehyde. Flow cytometric analysis was always performed with a FACS Calibur (BD Biosciences, Heidelberg, Germany) equipped with an argon laser. |
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